Stimulation of DNA Replication from the Polyomavirus Origin by PCAF and GCN5 Acetyltransferases: Acetylation of Large T Antigen

Abstract

The PCAF and GCN5 acetyltransferases, but not p300 or CBP, stimulate DNA replication when tethered near the polyomavirus origin. Replication stimulation by PCAF and GCN5 is blocked by mutational inactivation of their acetyltransferase domains but not by deletion of sequences that bind p300 or CBP. Acetylation of histones near the polyomavirus origin assembled into chromatin in vivo is not detectably altered by expression of these acetyltransferases. PCAF and GCN5 interact with polyomavirus large T antigen in vivo, PCAF acetylates large T antigen in vitro, and large T-antigen acetylation in vivo is dependent upon the integrity of the PCAF acetyltransferase domain. These data suggest replication stimulation occurs through recruitment of large T antigen to the origin and acetylation by PCAF or GCN5.Stimulation of DNA replication by auxiliary sequence elements located in cis to origins that function in eukaryotic cells was first observed with murine polyomavirus (Py) (28) and has since been documented for many other viruses. These auxiliary sequence elements bind proteins postulated to recruit and/or activate additional proteins involved directly in replication or to modify origin sequences and chromatin structures or their intranuclear localization (9, 19, 21, 22, 38-40, 48, 60, 63, 64, 72, 99, 100, 106, 108, 113; for reviews, see references 26 and 74). Much can be learned about the control of DNA replication from studying these proteins and their functions.The Py enhancer PEA1 and PEA3 sites are particularly important for stimulating Py DNA replication (18,20,39,48,69,71,75,83,90,91,105). Jun, a member of the AP1 (PEA1) complex, recruits Py large T antigen (PyLT) to the origin to stimulate DNA unwinding, particularly at early times after infection when PyLT is limiting (39,48,69,71,75,91). The AP1 complex and ets family proteins (that bind the PEA3 site) as well as Gal4VP16, NF-B, E1a, Sp1, and p53, which also can stimulate Py DNA replication (8,10,11,37,46,53,74,77,111; reviewed in references 26 and 74), interact with p300/CBP (2,(4)(5)(6)(7)31,36,50,57,61,65,68,82,101,116), PCAF, and GCN5 (15,67,107,110) and other coactivators that acetylate histones and nonhistone proteins involved in transcription, including HMG17, HMGI(Y), E2Fs, p53, c-Jun (109), MyoD, YY1, Tat, TF II E, TF II F, and TF I 68 (17,92,96). Acetylation regulates these proteins' functions and interactions with other proteins (17,55,92,96,97).Proteins directly involved in DNA replication also interact with acetyltransferases, including PyLT, which interacts with p300/CBP (23, 76), MCM2 and ORC1 (which interact with acetyltransferase HBO1 [14,44]), and MCM3, whose acetylation affects DNA replication (103). Also, acetyltransferases are recruited to double-stranded DNA breaks to facilitate DNA repair (13,45,70; for a review see reference 17). However, neither the specific roles for histone acetylation in these processes nor the proteins that catalyze them have been established.Here we demonstrate that the PCAF and GCN5 acetyltransferases, when tethe...

Eukaryotic MCM Proteins: Beyond Replication Initiation

“…A similar effect is observed if the Gcn5 histone acetyltransferase (HAT) is tethered next to an origin (329). Interestingly, tethering of PCAF/ Gcn5 HAT adjacent to the polyomavirus replication origin also stimulates replication in animal cells (342). Early studies suggested that regions of the chromatin associated with MCM proteins are more susceptible to nuclease digestion, indicating that these domains may be less tightly compacted (118,262).…”

“…As described above, the histone aceyltransferase PCAF/Gcn5 is reported to stimulate replication from a viral origin by contributing to the acetylation of polyomavirus T antigen, a viral helicase similar to MCMs (342). This suggests that the interaction of MCMs with other HATs such as Hbo1 may result in MCM acetylation.…”

“…Overexpression of a member of the Gcn5-containing SAGA complex partially suppresses the phenotypes associated with the mcm5 allele (70). This could indicate direct interactions such as those observed with polyomavirus large T antigen (342). Alternatively, MCMs may influence, or be sensitive to, the histone modifications mediated by this complex.…”

“…In addition, HATs are not limited to acetylating histones. The PCAF and Gcn5 HATs that stimulate polyomavirus replication do so by acetylating the polyomavirus large T antigen, which is required for origin binding and replication (342); in human cells, Mcm3 is also acetylated (see "Modifications of MCM" below). Thus, Hbo1 might directly modify MCMs.…”

Regulation of SV40 large T-antigen stability by reversible acetylation

“…The polyomavirus large T-Ag (PyLT) has been reported to be acetylated by PCAF and GCN5 (Xie et al, 2002). The acetylation of the PyLT is thought to have a role in stimulating DNA replication.…”